1. Field of the Invention
The present invention relates to a confocal microscope and a method of generating a three-dimensional image using a confocal microscope.
2. Related Background Art
A confocal microscope mainly comprises a laser light source, a dichroic mirror, a two-dimensional scanning unit for two-dimensionally scanning excitation light, an objective lens, a focusing lens, a pinhole formed on the focal plane of the focusing lens, and a photodetector.
Excitation light emitted by the laser light source is reflected by the dichroic mirror, and is brought into focus on a specimen via the two-dimensional scanning unit and the objective lens. Fluorescence produced by the specimen upon irradiation of the excitation light is transmitted through the dichroic mirror via the objective lens and the two-dimensional scanning unit. The fluorescence transmitted through the dichroic mirror is brought into focus on the pinhole set in the optical path by the focusing lens. At this time, only fluorescent components originating from the focal point of the excitation light pass through the pinhole, and are received by a detection device. The detection device outputs an electrical signal corresponding to the amount of received light. This signal and scanning position information are subjected to predetermined processing using a computer to obtain a slice image (a fluorescent image or a reflection image) of the specimen.
FIG. 7 is an explanatory view of the method of generating a three-dimensional image using the conventional confocal microscope.
By changing the distance (focal length) between the objective lens and a specimen, slice images of a plurality of planes E1 to E4 perpendicular to an optical axis L10 are acquired at the individual focal points. A three-dimensional image is generated by interpolating upper and lower slice images using a computer, and the generated image is displayed. This technique is disclosed in U.S. Pat. No. Re. 34,214.
FIG. 8 is an explanatory view of the method of obtaining an inclined slice image from the slice images shown in FIG. 7, and FIG. 9 is an explanatory view of the method of generating a three-dimensional image from the inclined slice images.
An image on a plane perpendicular to the optical axis L10 is projected onto planes F1 and F2 which are perpendicular to axes L20 and L30 which are slightly inclined from the optical axis L10 and are symmetrical about the optical axis L10. Images F11 to F14 and F21 to F24 obtained by projection at the individual focal points are sequentially overlaid on each other while being shifted in accordance with their inclinations. With this processing, images viewed from different directions are generated. These images are juxtaposed to display a three-dimensional image using stereoscopic viewing or the like, or the images are overlaid on each other while changing their colors, and a three-dimensional image is generated using color spectacles.
On the other hand, in place of projecting images, a pseudo three-dimensional image can be displayed by overlaying images obtained at the individual focal points while shifting them.
With the method shown in FIG. 7, a three-dimensional image that suffers less distortion can be generated, and can be easily rotated. However, since the generation and rotation of the three-dimensional image require many calculations, an expensive computer is required to obtain a three-dimensional image at high speed.
In the method shown in FIGS. 8 and 9, calculations for projection must be done by a computer, and it is time-consuming to generate a three-dimensional image.
Furthermore, in the method that does not perform any projection, the calculations of a computer are facilitated, and a three-dimensional image can be generated at high speed. However, since a three-dimensional image is directly generated on the basis of images obtained by scanning on planes perpendicular to the optical axis, spatial distortion is produced.